Effects of isoinertial training on muscle power, endurance, isometric strength, and balance: randomized clinical trial in patients with post-ACL reconstruction

Abstract

Background

ACL reconstruction surgery leads to temporary limitations in knee movement due to weakness in the quadriceps, swelling, and stiffness. Effective therapy is necessary to regain strength and functionality. While flywheel resistance training enhances strength and eccentric loading, there is limited knowledge about its effectiveness in conjunction with conventional rehabilitation methods. This study evaluates the impact of both conventional rehabilitation and isoinertial therapy on muscle power, endurance, isometric strength, and balance in patients following ACL repair.

Methods

A total of 96 out of 136 patients aged 18 to 45 who had been randomly assigned to groups were included 3 weeks post-procedure. Over 6 weeks, 47 patients in group A (n = 47) participated in both isoinertial training and conventional therapy protocals while in group B (n = 49) followed a conventional rehabilitation protocol for just 30 min daily. To assess the patients’ muscle power, endurance, isometric strength, and balance, a flywheel ergometer (D11 Plus; Desmotec, Biella, Italy) was utilized both before and after the 6-week treatment period.

Results

Group A had statistically significant increases in eccentric muscular power (p = 0.0001), whereas there was no noticeable difference between the two groups in isometric strength, balance, or concentric power (p = 0.731, p = 0.786, and p = 0.815, respectively).

Conclusion

The finding indicates that isometric strength, balance, and both concentric and eccentric muscular power were successfully improved by both 6-week interventions: traditional rehabilitation alone and in conjunction with isoinertial training. But when compared to traditional therapy alone, the isoinertial training group showed better gains in muscular endurance. There were no discernible variations in the groups’ isometric strength or balance results. The study also emphasizes how isoinertial training can effectively increase eccentric power.

Trial registration.

The ethics approval number is LPU/IEC/2018/01/09. Registered in the Clinical Trials Registry of India under CTRI/2019/06/019858 dated 23/09/2021.

Background

The knee is one of the most injured joints in the human body. Participating in sport greatly increases the risk of injury to the knee. In recent studies, 40% of all ligamentous injuries to the knee are caused by anterior cruciate ligament (ACL) injuries, with 70% of ACL injuries occurring during athletic activity.[1] ACL injuries are widespread in many sports, including football, basketball, skiing, netball, volleyball, and rugby, because these sports involve jumping, cutting, and decelerating, which are the top causes of ACL injuries.[2] An ACL damage raises the risk of further injuries by causing recurring periods of instability at the knee joint. However, ACL rupture is no longer seen as a career-threatening injury due to advancements in surgery and rehabilitation. Study shows 89% of professional football players resume their prior level of play following a knee injury.[3, 4]

The assessment of ACL surgery, outcomes, and rehabilitation has been explored extensively in various studies over the years.[5] Research indicates that the main focal points are injury prevention and perturbation training. After undergoing ACL surgery, rehabilitation commonly incorporates perturbation training, which may consist of dynamic proprioceptive exercises and activities tailored to specific sports, aiming to lower the chance of recurrence and possibly tackle the fundamental reason for the injury.[6, 7]

Rehabilitation following ACL replacement is continuing to advance rapidly. A review of recent research indicates that functional knee bracing and perturbation training programs are advantageous in rehabilitation programs that occur prior to, during, and after surgery. The necessity for a criteria-based approach to progress through the final stages of ACL reconstruction rehabilitation, as well as the return to sports, is also highlighted.[8] Eccentric overload exercise has been shown in numerous studies to be highly beneficial for rehabilitation after ACL repair surgery.[9]

Isoinertial training is a “newly developed technique that is becoming very popular worldwide for patient rehabilitation.” The isoinertial modality provides an additional eccentric load. Originally, flywheel-based isoinertial training was designed to maintain the health of astronauts’ muscles while in space.[10] Instead of relying on gravity-dependent weights, it utilizes isoinertial technology, which facilitates maximal concentric and eccentric muscle activities through brief periods of eccentric overload.[11] Research has shown that flywheel resistance training increases eccentric muscle loads and boosts strength. [12] In healthy individuals, isoinertial exercise leads to greater peripheral neuronal adaptations and muscle growth compared to weight-loaded resistance training, likely due to the added eccentric overload.[13]

Numerous rehabilitation techniques have been shown to effectively support patients recovering from anterior cruciate ligament reconstruction surgery, as indicated by previous research studies. Isoinertial concentric-eccentric maximum exercise has been identified as a viable therapeutic approach within various post-surgical rehabilitation protocols since it does not lead to significant muscle damage or clinically relevant delayed onset muscular soreness (DOMS). However, for optimal outcomes, this treatment approach should not be used in isolation. There is a scarcity of case studies exploring the combined effects of isoinertial training for eccentric strengthening in the context of ACL repair procedures. Therefore, the objective of this study is to investigate how postoperative patients who have undergone ACL reconstruction surgery perform concerning muscle power, endurance, isometric strength, and balance when they participate in an isoinertial training regimen in combination with a traditional rehabilitation program or solely follow the conventional rehabilitation protocol.

Methodology

The study used an interventional experimental design in accordance with CONSORT (Consolidated Standards of Reporting Trials) guidelines as part of a randomized controlled trial to evaluate the effectiveness of isoinertial eccentric overload strengthening in addition to conventional rehabilitation techniques in patients having ACL reconstruction surgery. From July 2020 to September 2023, Fortis Hospital in Ludhiana, Punjab, India, served as the site of the study. Clinicaltrials.gov registered the study (CTRI/2021/09/036933) and the Institutional Human Ethical Committee granted ethical approval. Written and verbal agreement was obtained from each patient, and the Indian Medical Research Council’s Good Clinical Practice and the World Medical Association’s 2013 Declaration of Helsinki’s ethical guidelines were closely adhered to.

The sample size of 95 patients was determined by predicting a minimum of 82 participants using a two-tailed test, α = 0.05, power (1 − β) = 0.80, and effect size = 0.25, using G*Power 3.1 software. We also expanded the sample size to 96 to accommodate for a 15% dropout rate. Patients were randomly divided into two groups, group A and group B. A lottery approach was used for randomization, where an impartial administrator who was not participating in the trial drew sealed opaque envelopes. All the time, allocation concealment was kept intact. The Consolidated Standards of Reporting Trials (CONSORT) is shown in Fig. 1.

Fig. 1.

CONSORT diagram showing the flow of participants through each stage of a randomized trial

Group A received both isoinertial strengthening and the conventional rehabilitation treatment, whereas Group B received only the conventional rehabilitation protocol. Patients between the ages of 18 and 45 who had undergone ACL reconstruction surgery 3 weeks prior and who had been treated by the same surgeon with a semi-tendinosis gracilis graft were eligible. Patients who had previously undergone ACL reconstruction surgery, had previously undergone knee surgery, refused to sign the consent form, had physical limitations that affected their gait or lower limb activity, were unable to perform isoinertial training, or did not understand English, Hindi, or Punjabi were excluded.

One hundred thirty-six patients at Fortis Hospital who had ACL reconstruction surgery were screened for the study; 96 of them satisfied the selection requirements. Group assignment was concealed from outcome assessors. Because of the nature of the intervention, therapists who provided treatment were not included in outcome assessments but could not be blinded. Using a flywheel ergometer (D11 Plus; Desmotec, Biella, Italy), the muscle power, endurance, isometric strength, and balance of each patient were evaluated. Assessments were performed at baseline and following the intervention period, which lasted 6 weeks for 60 min in group A and 30 min in group B, respectively.

Flywheel ergometer (D11 Plus; Desmotec, Biella, Italy): Peak force was measured using an isoinertial device (D11 full, Desmotec, Biella, Italy) to conduct the lower extremity isometric strength test (ISOMET). A strap that had one end fastened to the gadget and the other to the participant’s vest served as the link between the two. To prevent the respondent from moving up, the strap was tightened. Two contact panels of the Desmotec device are linked to a computer running the software (D. Soft, Desmotec, Biella, Italy). The participant places his hands on his hips while standing in a semi-squat stance with his flexion at a 100-degree angle. The subject applies pressure to the plates for 10 s at the sign, which is the maximum voluntary isometric contraction. The participant’s force is measured by the contact panels and recorded on the computer. Thus, isometric strength and endurance were evaluated along with eccentric and concentric muscular power. For 10 s, the patient stands on the platform with loaded plates in a semi-squat position with his hands on his hips and flexion at a 100-degree angle. Any deviation from the zero on either the left or right side is noted, and the percentage of deviation is thus tracked.[14]

Interventions

Group A

The experimental group consisted of 47 subjects who received an additional 30-min isoinertial strengthening protocol twice a week for 6 weeks. This was done in addition to the conventional rehabilitation protocol (Table 1), which was given every day for 6 weeks until phase 2 and a half. The treatment was given for 30 min every day, 6 days a week.[15]

Table 1.

Description of conventional rehabilitation protocol

Phases in weeks	Criteria for progression	Details of the protocol
Phase 1 Week 1	–	• Control of pain and inflammation (i.e., through cryotherapy and exercises) • Obtain ROM of 0–90*, emphasizing achievement of full extension (i.e., through CPM and exercises: patellar mobilization in all directions, heel slides and leg elevation with a pillow under the heel) • Regain muscle control, with safe isometric and isotonic OC (ROM 90*–40*) and CC (ROM 0*–60*) strength exercises without additional weight (i.e., SLR, mini squads, shifting body weight) • Improve gait pattern. If pain is tolerated, aim at walking without crutches from day 4. Sufficient neuromuscular control and a non-limping gait pattern are criteria for walking without crutches
Phase 2 Week 2 to week 9	• Pain knee is equal to previous week or less (VAS-score pain) • Minimal swelling (measurement with measuring tape) • Full extension and 90 flexion are possible (ROM goniometer) • Good patellar mobility compared with contralateral side • Sufficient quadriceps control to perform a mini squad 0–30 and SLR in multiple directions • Ability to walk independently with or without crutches	• Apply cryotherapy in case of pain or swelling (if necessary, after each therapy session) • Work toward full ROM (maintain full extension, 120* flexion from week 2 and 130* flexion from week 5) with remaining attention for good patellar mobility • Walking without crutches from day 4 to 10. Normalize gait pattern with walking exercises (treadmill from week 3 and jogging in a straight line from week 8) • Isometric and isotonic strength training increasing in intensity (quadriceps, hamstring, gastrocnemius, and soleus), with increasing ROM for OC and CC exercises without extra weight. For OC exercises: weeks 2, 3, and 4 from 90* to 40*, afterward 10* toward extension to be added every week. For CC exercises: weeks 2–7 from 0* to 60* and from week 8 from 0* to 90* • Start neuromuscular training by slowly increasing from static stability to dynamic stability. Work toward confidence on the vestibular and somatosensory system for balance, with increasing surface instability and decreasing visual input • Start from week 3 with cycling on an ergometer and swimming • Start from week 4 with stepping on a stair-stepping machine • Start from week 8 with outdoor cycling Caution: act adequately in case of persisting pain, inflammation, or limited ROM. There is a risk of developing arthrofibrosis (in case of doubt consult the orthopedic surgeon)
Phase 3 Week 9 to week 16	• Minimal pain and swelling (VAS-score pain, measurement of knee swelling with measuring tape) • Full extension and at least 130* flexion possible (ROM goniometer) • Normal gait pattern • Exercises of previous week are carried out properly • Administer the IKDC questionnaire	• Obtaining and maintaining full ROM • Optimizing muscle strength and endurance. Add increasing weights from week 9 both for OC and CC exercises • Neuromuscular training with increasing emphasis on dynamic stability and plyometric exercises, slowly increasing duration and speed. Start with two-legged jumping and work slowly toward one-legged jumping. Normalize running with outdoor jogging from week 13
Phase 4 Week 16 to week 22	• No pain or swelling in the knee (VAS-score pain, measuring knee swelling with measuring tape) • Full flexion and extension of the knee (ROM goniometer) • Administer the IKDC questionnaire again • Quadriceps and hamstring strength > 75% compared to the contralateral side. Difference in hamstring/quadriceps strength ratio is < 15% compared to the contralateral side (optional isokinetic strength testing of knee flexors and extensors at 180 per second) • Hop tests > 75% compared to the contralateral side • Exercises of previous week are carried out properly	• Maximizing muscle endurance and strength • Maximizing neuromuscular control with emphasis on jumping, agility training, and sport-specific tasks. Variations in running, turning, and cutting maneuvers are allowed. Duration and speed to be increased and maximized
–	Criteria for returning to sports • No pain or swelling (VAS-score pain, measuring knee swelling with measurement tape) • Full flexion and extension of the knee is possible (ROM goniometer) • Quadriceps and hamstring strength > 85% compared to the contralateral side. Difference in hamstring/quadriceps strength ratio is < 15% compared to the contralateral side (optional isokinetic strength testing of knee flexors and extensors at 60*, 180*, and 300* per second and an endurance test at 180* per second) • Hop tests > 85% compared to the contralateral side • Exercises of previous week are carried out properly, and the patient tolerates sport-specific activities and agility training with maximal duration and speed • Administer the IKDC questionnaire again	Exercises of previous week are carried out properly, and the patient tolerates sport-specific activities and agility training with maximal duration and speed

This protocol used a flywheel ergometer (D11 Plus; Desmotec, Biella, Italy) to assess maximal power peak, muscle endurance, balance shift, total work done, and maximal isometric force. Using a flywheel ergometer (D11 Plus; Desmotec, Biella, Italy), a half-squat exercise was used to accomplish eccentric overload training. The regimen of lunges and squats on the flywheel ergometer’s pressure plate platform in three sets of ten repetitions each, done twice a week, with a 1-min passive recovery period in between (see Fig. 2). An investigator qualitatively assessed each repetition, providing the individuals with kinematic feedback and strongly motivating them to execute to the best of their abilities within the intended power zone. The patients were told to control the eccentric phase until the knee reached about 90° of flexion and then execute the concentric phase as quickly as possible. During EOL exercise, each patient will be given the cumulative load listed below: Two disks were identified: one large (diameter 0.285 m; mass 1.9 kg; inertia 0.02 kg·m²) and one medium (diameter 0.240 m; mass 1.1 kg; inertia 0.008 kg·m²). The ergometer’s (D11 Plus) calculated inertia is 0.0011 kg·m² (refer Table 1).[16]

Fig. 2.

Group A on regimen on the flywheel ergometer with conventional rehabilitation protocol (A1 to A3)

Group B

Forty-nine patients in group B received conventional rehabilitation protocol for a total of 6 weeks, 30 min a day, 6 days a week, until week 6 of phase 2 and a half.[15] Table 1 provides information on the interventional program, shown in Fig. 3.

Fig. 3.

Group B on conventional rehabilitation protocol (B1 to B4)

Statistical analysis

The SPSS software was used to analyze the data. The independent t-test was used to compare the baseline characteristics of the groups. The effects of the interventions within the groups were evaluated using Student’s t-test for all outcome measures. The 95% CI was computed with alpha set at 0.05. The independent t-test was used to determine whether a significant difference was found.

Results

The mean age (years) of the patients in groups A and B of this study was 29.94 ± 6.78 and 30.20 ± 7.45, respectively (p = 0.832), suggesting that there is no discernible age difference between the groups (see Table 2). The baseline values of muscle power, endurance, isometric strength, and balance, together with the patients’ demographic characteristics (age, height, and weight), were compared and found to be statistically non-significantly different.

Table 2.

The demographic characteristics of age, height, weight, and gender

Parameter	Group A (n = 47)	Group B (n = 49)	t value	p value
Age (years)	29.94 ± 6.78	30.20 ± 7.45	− 0.179	0.858
Height (cm)	170.65 ± 4.63	172.13 ± 5.54	− 1.423	0.158
Weight (kg)	78.28 ± 9.94	77.84 ± 10.44	0.212	0.833
Gender (M/F)	36 M/11 F	39 M/10 F	X2 = 0.145	0.703

cm centimeter, kg kilogram, M male, F female, X² chi-square.

Table 3 explains how postoperative patients who had ACL repair surgery compared their mean values for muscle power, endurance, isometric strength, and balance before and after the intervention within the two groups. The findings of the analysis indicated that both groups A and B had statistically improved muscle power (both concentric and eccentric), isometric strength, and balance (p < 0.05). In contrast, there was no statistically significant difference in muscle endurance between group B at two separate intervals (0 week and 6th week) at p > 0.05, as shown in Fig. 4.

Table 3.

Comparison of mean values of muscle power, endurance, isometric strength, and balance before and after the intervention within the two groups

Parameters	Group A (experimental group)				Group B (control group)
	Week 0 Mean ± S.D	Week 6 Mean ± S.D	t value	p value	Week 0 Mean ± S.D	Week 6 Mean ± S.D	t value	p value
Muscle power concentric (W)	79.00 ± 19.35	173.53 ± 49.91	16.42	< 0.0001*	80.61 ± 25.78	172.51 ± 50.32	18.109	< 0.0001*
Muscle power eccentric (W)	68.19 ± 15.37	178.40 ± 41.96	25.88	< 0.0001*	89.20 ± 18.57	163.10 ± 36.11	24.780	< 0.0001*
Endurance (s)	10.98 ± 3.28	14.85 ± 3.87	9.499	< 0.0001*	11.10 ± 2.39	11.65 ± 2.26	1.826	0.0740
Isometric strength (kg)	166.70 ± 33.15	216.33 ± 36.6	7.210	< 0.0001*	161.30 ± 41.08	211.93 ± 42.71	22.630	< 0.0001*
Balance (%)	6.54 ± 2.71	3.23 ± 2.10	7.313	< 0.0001*	7.16 ± 2.08	4.00 ± 2.50	7.210	< 0.0001*

*Significant (p value < 0.05 considered statistically significant)

Fig. 4.

Graphical representation of comparison of mean values of muscle power, endurance, isometric strength, and balance

Furthermore, there was no statistically significant difference between groups A and B (p > 0.05) based on the study of the improvement of mean values of muscle power (concentric), isometric strength, and balance (refer Table 4). The improvement of the mean value of muscle power (eccentric) in group A, however, showed a statistically significant difference (p < 0.0001).

Table 4.

Comparison of improvement in the mean values of muscle power, isometric strength, and balance

Parameters at 0 week vs 6 weeks	Group A Mean ± S.D	Group B Mean ± S.D	t value	p value
Muscle power concentric (W)	94.53 ± 39.47	91.90 ± 35.52	0.344	0.731
Muscle power eccentric (W)	110.21 ± 29.19	73.90 ± 20.8	7.032	< 0.001*
Isometric strength (kg)	49.63 ± 19.88	50.62 ± 15.66	0.271	0.786
Balance (%)	− 3.311 ± 3.10	− 3.16 ± 3.07	0.233	0.815

*Significant

W watt, kg kilogram, s seconds.

Note: Statistical tests: The independent t-test was used for comparisons between groups, while the paired t-test was used for comparisons within groups.

W watt, kg kilogram, S.D standard deviation.

Discussion

In this research, patients who underwent ACL reconstruction surgery were monitored for 6 weeks to assess the effects of combining an isoinertial training program with a conventional rehabilitation program compared to a conventional rehabilitation program alone on muscle power, endurance, isometric strength, and balance. While the group that followed the isoinertial training alongside conventional rehabilitation showed improvements in endurance, no such advancements were noted in the group that received only the conventional rehabilitation. Both patient groups those who engaged in isoinertial training plus conventional rehabilitation and those who participated in only conventional rehabilitation experienced enhancements in muscle power during both concentric and eccentric phases, as well as improvements in isometric strength and balance. The study did not consider participants’ dietary intake. Although participants were advised to maintain their usual eating habits, future studies should incorporate the evaluation and adjustment of diet, as nutritional intake may influence recovery.

After surgery for ACL reconstruction, muscle power that includes both concentric and eccentric components is essential for recovery. Muscle shortening and concentration are necessary for restoring joint stability and functional strength. Muscular lengthening or eccentric contractions are essential for regulated movement during tasks like stair climbing, improving muscular flexibility, and accelerating tendon repair. In the current study, both group A and group B showed increases in muscle power during the concentric and eccentric phases. These results are in line with those published by Lorenz and Reiman[18] and Gokeler et al.[17]

The mean eccentric power improvement showed that conventional therapy plus isoinertial training is more effective than conventional rehabilitation alone. In the study by de Hoyo et al.,[19] it was found that flywheel exercises have a higher eccentric load because the subject performs a high velocity movement (usually maximal) during the positive (extension) phase of a squat, while the subject must break the load accumulated during the negative (flexion) phase. The main benefit of EOL is therefore associated with an enchained mechanical load. A positive transfer in motor unit recruitment, force, and power output may have been ensured by the authors’ earlier research, which hypothesized that a high eccentric load may have better stimulated higher order motor units (which need the use of high load).[16, 20]

Muscle endurance is essential for reestablishing regular gait patterns, strengthening joints, and increasing general functional ability, all of which help patients carry out everyday tasks with less discomfort. Participants in the current study showed a significant increase in muscle endurance, rising from a mean value of 10.98 to 14.85, when they received isoinertial exercise in addition to traditional therapy. On the other hand, the group that only underwent conventional rehabilitation protocol showed no discernible improvement. The improved ability of eccentric muscle activities, a crucial part of isoinertial training, to generate torque may be the reason for the better results seen in the combined intervention group.[21] However, maximal electromyographic (EMG) activity, which may be lower than in concentric activities, suggests that neural activation is not greater with eccentric muscle action. Conversely, eccentric muscular actions may improve protein synthesis in muscles and contractile tissues by increasing muscle tension. The first week of consistent training will reduce the soreness following eccentric activity.[22] Muscle endurance is increased because many natural movement patterns involve eccentric muscle activation, particularly when the stretch–shortening cycle is employed.

Rehabilitation following ACL reconstruction should include both balance and isometric strength training. With little mechanical strain on the knee, isometric exercises which include static muscle contractions without joint movement are useful for improving joint stability, reducing muscle atrophy, and regaining periarticular strength. They also help to enhance neuromuscular control, which is necessary for motor coordination and the avoidance of compensatory movement patterns. Training for balance is equally important since it improves joint position awareness and proprioception, which lowers the chance of falls and re-injuries. The results of the current study showed that the isometric strength and balance improvements of the two intervention groups those undergoing conventional rehabilitation and those receiving isoinertial training were equivalent. Regarding effectiveness, there were no discernible variations between the two methods.

Direct comparisons with the existing body of research are challenging due to the limited number of studies exploring the impact of these therapies on isometric strength and balance in postoperative ACL populations. The improvement noted in group A may be attributed to isoinertial training, which employs flywheel devices to provide varying resistance during both concentric and eccentric phases. This training modality’s close resemblance to dynamic loading and functional movement patterns may lead to greater enhancements in isometric strength and balance. Additionally, isometric exercises focused on joint stabilization and the development of static strength were included in the typical rehabilitation protocol, which may account for the neuromuscular improvements observed in this group. The distinct yet complementary effects of the various training methods likely contribute to the observed enhancements in strength and balance in both groups.

This study has few limitations. The nature of the intervention prevented therapists from being blind to group assignments, which could have led to performance bias. Further restricting the capacity to separate the effects of each intervention was the inability to directly compare the isoinertial training protocol with the traditional rehabilitation treatment alone. Furthermore, it is more difficult to evaluate long-term results and the longevity of the noted changes when there is no long-term follow-up. The effectiveness of isoinertial eccentric-oriented training in conjunction with a traditional rehabilitation strategy was supported by this study despite these limitations. The small sample size prevented subgroup analysis by age, which could have impacted statistical power. It is important to investigate age-specific outcomes in future research using larger cohorts. The study’s future scope entails performing long-term follow-up evaluations 4 to 5 months after rehabilitation to look at the long-term effects on activity levels and lifestyle modifications. This prolonged observation period would yield important information about the long-term impacts of the rehabilitation procedures and how they affect the patients’ everyday routines and way of life in general.

Conclusion

This research indicated that both treatment protocols—combining isoinertial training with conventional rehabilitation and using conventional rehabilitation alone—were successful in improving muscle power during both concentric and eccentric phases, as well as isometric strength and balance over a duration of 6 weeks. Conversely, group A that underwent isoinertial training in conjunction with conventional rehabilitation demonstrated enhancements in endurance, while group B, which only participated in conventional rehabilitation, did not exhibit any progress. Further investigations revealed that an isoinertial training program combined with a traditional rehabilitation approach can significantly boost power in the eccentric phase of muscle contraction.

Abbreviations

ACL

Anterior Cruciate Ligament

CC

Close Chain

CONSORT

Consolidated Standards of Reporting Trials

DOMS

Delayed Onset Muscular Soreness

EMG

Electromyographic

IKDC

International Knee Documentation Committee

ISOMET

Isometric Strength Test

OC

Open Chain

ROM

Range of Motion

SLR

Straight Leg Raising

VAS

Visual Analogue Scale

Authors’ contributions

RCP contributed to conceptualization, data curation, formal analysis, investigation, methodology, supervision, validation, writing—original draft, and writing—review and editing; SG did the data curation, formal analysis, methodology, investigation, validation, and writing—review and editing; SM contributed to data curation, formal analysis, investigation, methodology, validation, and writing—review and editing; AY contributed to data curation, formal analysis, investigation, methodology, validation, and writing—review and editing; SM uo.

Funding

The authors did not receive funding for this article’s research, writing, and publication.

Data availability

The data will be available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The study was approved by the Institutional Review Board (IRB). The ethics approval number is LPU/IEC/2018/01/09. It was filed in the Clinical Trials Registry of India under CTRI/2019/06/019858. Written informed consent was obtained from all subjects involved in the study to publish this article.

Consent for publication

The participants were provided with informed consent before publishing any information.

Competing interests

The authors disclosed no potential conflicts of interest regarding this article’s research, authorship, and publishing.